The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
DNA methylation is one of the most intensely studied epigenetic modifications in mammals and refers to the addition of a methyl (CH3) group to a cytosine (C) or adenine nucleotides. This methyl group may be added to the fifth carbon atom of the cytosine base or the sixth nitrogen atom of the adenine base.
DNA methylation plays a role in gene regulation in animal cells. Not only is there a correlation between active gene transcription and hypo-methylation, but also transfection experiments show that the presence of methyl moieties inhibits gene expression in vivo. Furthermore, gene activation can be induced by treatment of cells with 5-azacytidine, a potent demethylating agent. Methylation appears to influence gene expression by affecting the interactions of DNA with both chromatin proteins and specific transcription factors. Although methylation patterns are very stable in somatic cells, the early embryo is characterised by large alterations in DNA methylation.
DNA methylation is therefore vital to healthy growth and development and is linked to various processes such as genomic imprinting, carcinogenesis and the suppression of repetitive elements. It also enables the expression of retroviral genes to be suppressed, along with other potentially dangerous sequences of DNA that have entered and may damage the host. In addition, DNA methylation plays an important role in the development of cancer and is a key regulator of gene transcription. Studies have shown that genes with a promoter region that contains a high concentration of 5-methylcytosine are transcriptionally silent.
Between 60% and 90% of all CpGs are methylated in mammals. Methylated cytosine residues spontaneously deaminate to form T residues over time; hence methylated CpG dinucleotides steadily deaminate to TpG dinucleotides, which is evidenced by the under-representation of CpG dinucleotides in the human genome (they occur at only 21% of the expected frequency). CpGs are often grouped in clusters called CpG islands, which are typically present in the 5′ regulatory regions of many genes.
With growing evidence of the diagnostic utility of monitoring DNA methylation levels, means for reliably and accurately assessing DNA methylation is becoming increasingly important. Currently, methylation-specific PCR is a commonly used method for detecting methylated DNA in bisulphite-converted DNA. In this method, PCR oligonucleotide primers interrogate methylated cytosine residues in cytosine-phosphodiester-guanidine [CpG] sites. MethyLight PCR is a real-time PCR variation which, in addition to methylation specific primers, also uses a 5′-3′ hydrolysis probe for interrogation of methylated CpG sites, thereby enabling quantification.
More recently, RNA has also been shown to contain methylated cytosine residues, as well as methylated adenine residues (Liu and Jia, 2014; J Genet Genomics. 41(1):21-33). Although the biological role of methylated cytosine in RNA is unclear, it is an abundant modification in mRNA, suggesting that it might be an RNA epigenetic marker. In work leading up to the present invention it has been determined that in the context of some diagnostic applications which are based on screening for a change in the methylation pattern of a given gene, the accuracy of the diagnostic result is significantly reduced where partial methylation exists across a CpG-rich target region of interest. This is due to the fact that a commonly used methylation specific PCR is based on oligonucleotides requiring all targeted CpG sites to be methylated. For example, a probe-based method, such as MethyLight, requires that all interrogated CpG sites are methylated in order for the probe to hybridise for the successful detection of a given methylated DNA or RNA target. Where one or more CpG sites are not methylated, the probe cannot bind, thereby skewing the results that are obtained and significantly reducing diagnostic sensitivity. For example, in one aspect of the present invention, it has been determined that methylation of the promoter region of the IKZF1 gene occurs with high frequency in colorectal cancer tissues and that the detection of methylated IKZF1 DNA in cell free DNA which is present in the blood indicates the presence of colorectal cancer. Further studies, however, have demonstrated that certain colorectal cancer patients contain circulating tumour-derived IKZF1 DNA where not all the targeted CpG sites are methylated. Thus, an oligonucleotide, such as a hydrolysis probe designed to span methylated CpG sites within the IKZF1 DNA, does not allow detection of partially-methylated IKZF1 DNA. Accordingly colorectal cancer patients with a partially methylated IKZF1 DNA will consequently be reported as negative.
Accordingly, there is a need to develop improved methods that enable accurate and sensitive detection of DNA or RNA methylation, thereby improving the sensitivity of the applications for DNA or RNA methylation analysis, such as the diagnosis or monitoring of neoplastic disease. In still further work, it has been determined that the problem of false negative results can be reduced or eliminated via the use of one or more probes and/or primers which are designed to collectively detect at least two differing methylation patterns within a given DNA or RNA region of interest.